Apoptosis/Necrosis

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Apoptosis, often called programmed cell death, is a carefully regulated process that is part of normal development and homeostasis. Apoptosis is morphologically and biochemically distinct from necrosis, which is conversely called accidental cell death. Dysregulation of apoptosis is implicated in disease states such as cancer, autoimmune disease and degenerative conditions.

Apoptosis consists of an orderly sequence of events characterized by cell shrinkage, increased cell permeability, changes in membrane asymmetry, chromatin condensation, DNA fragmentation, and cell blebbing. Finally, the apoptotic cells are removed through phagocytosis with minimal tissue disruption.No single parameter fully defines cell death in all systems, so it is advantageous to use several different apoptosis assays when studying apoptosis. The multiparametric nature of flow cytometry allows the measurement of several apoptotic traits in a single sample, making it a powerful tool to study the complexity of cell death.You can characterize apoptotic events in early, intermediate and late stages; let’s look at some of the most reliable apoptosis assays for routine use.

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Necrosis is an unprogrammed death of cells and living tissues, which does not follow a highly regulated intracellular program such as the apoptotic signal transduction pathway. necrotic cell death is not the result of one or two well-described signaling cascades but is the consequence of an extensive cross talk between various molecular events. Six characteristic morphologic patterns of necrosis are distinguished in pathology: coagulative necrosis, caseous necrosis, liquefactive necrosis, fat necrosis, fibrinoid necrosis, and gangrenous necrosis. Necrosis typically results in a loss of cell membrane integrity and an uncontrolled release of products of cell death into the extracellular space, which initiates an inflammatory response. Typical morphologic features of necrotic cell death, which occur in most but not in all eukaryotic cells, include mitochondrial swelling, lysosome rupture, and plasma membrane rupture. Necrosis can be induced by injury, infection, heat, cancer, infarction, toxins, and inflammation. Necrosis is accompanied by the release of special enzymes, which are stored by lysosomes and capable of digesting cell components.

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There are different way to detect apoptosis by flow cytometry.

•  Mitochondrial membrane potential with dyes( TMRE and JC-1). When a cell enters apoptosis, reduced fluorescence from these dyes.

•  Active caspase 3 will only be detected in apoptotic cells.

•  Phosphotidylserine residues are externalized during apoptosis, which are normally located on the internal surface of the plasma membrane,so only cells that have decided to die will be detected by Annexin V binding.

• Apoptotic cells become more permeable to a variety of DNA-binding dyes, such as ethidium bromide, 7-AAD , YO-PRO-1  and Hoechst 33342.

• As apoptosis progresses, DNA becomes fragmented. You can detect the fragmented DNA by an end-labelling technique or simply treating the cells with detergent to extract the DNA fragments and looking at DNA content. This is the widely used “Sub-G1 method” where apoptotic cells show DNA content below normal G1 cells.

 

Reference:

    

• http://flowbook.denovosoftware.com/chapter-9-cell-death-including-apoptosis

• Wlodkowic D., Skommer J., Darzynkiewicz Z. (2009) Flow Cytometry-Based Apoptosis Detection. In: Erhardt P., Toth A. (eds) Apoptosis. Methods in Molecular Biology (Methods and Protocols), vol 559. Humana Press, Totowa, NJ

• Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T. and Traganos, F. (1997). Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1–20.

• Darzynkiewicz, Z., Li, X. and Bedner, E. (2001). Use of flow and laser-scanning cytometry in analysis of cell death. Methods Cell Biol,.66, 69–109.

• Darzynkiewicz, Z., Huang, X., Okafuji, M. and King, M.A. (2004). Cytometric methods to detect apoptosis. Methods Cell Biol.75, 307–41.Scholar

• Wlodkowic, D., Skommer, J. and Darzynkiewicz, Z. (2008). SYTO probes in the cytometry of tumor cell death. Cytometry A 73, 496–507

• Leist, M. and Jaattela, M. (2001). Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol.2, 589–98.

• Kroemer, G. and Martin, S.J. (2005). Caspase-independent cell death. Nat. Med.11, 725–30.

• Blagosklonny, M.V. (2000). Cell death beyond apoptosis. Leukemia 14, 1502–8.

• Zhivotovsky, B. (2004). Apoptosis, necrosis and between. Cell Cycle 3, 64–6.

• Telford, W.G., Komoriya, A. and Packard, B.Z. (2004). Multiparametric analysis of apoptosis by flow and image cytometry. Methods Mol. Biol.263, 141–60.

• Castedo, M., Ferri, K., Roumier, T., Metivier, D., Zamzami, N. and Kroemer, G. (2002). Quantitation of mitochondrial alterations associated with apoptosis. J. Immunol. Methods 265, 39–47.

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